The renal thick ascending limb of Henle's loop (TALH) transports chloride actively against its electrochemical potential gradient. Its movement is inhibited by ouabain, the 'loop' diuretics such as furosemide and its movement appears to be coupled to the movement of sodium across the luminal membrane via a sodium-chloride cotransport system. Although sodium-chloride cotransport systems have been extensively described in mammalian epithelia as the small intestine, gall bladder and trachea by electrophysiological measurements, recently studies using plasma membrane vesicles have cast doubt on whether a direct coupling of sodium and chloride fluxes really exists or whether a 'loose' coupling via Na/H and Cl/OH exchange mechanisms occurs. Thus, this project proposes to investigate the mechanisms of sodium and chloride transport in the medullary TALH. The transport mechanisms for sodium and chloride entry into the cell will be defined by conducting flux studies in isolated TALH cells and in luminal plasma membrane vesicles prepared from these cells. These studies will investigate whether a dependency of sodium on chloride fluxes and conversely a dependency of chloride on sodium fluxes can be demonstrated. This is an essential criterium for the proof of a cotransport system. The characteristics of the sodium-chloride cotransport system will be studied including its localization within the cellular envelope, the anion and cation specificity, the sensitivity to a variety of diuretics and the role of potassium in the cotransport system. The stoichiometry of the cotransport system will be studied in order to elucidate the origin of the lumen-positive potential difference which exists in the intact tubule. Additionally, the kinetic parameters of the cotransport system will be measured in order to define quantitatively the molecular characteristics of the transport system. Through the results of this study the mechanisms and biochemical properties of the sodium-chloride cotransport system, and thus the process of urinary concentration, will be better understood.